The present invention relates to the production, by genetic engineering, of plasmids and bacterial strains containing the gene tfdA, or a gene essentially identical to tfdA, on a short, exactly characterizable DNA segment. The novel plasmids and microorganisms are especially well suited for the production of 2,4-D-monooxygenase, as well as for serving as starting compounds for the genetically engineered transfer of the 2,4-D-degrading properties of this enzyme to various organisms (including the thus-attainable 2,4-D-tolerance in genetically transformed plants).
2,4-D-Monooxygenase is an enzyme catalyzing in many 2,4-D-degrading organisms the first step in the metabolizing of 2,4-dichlorophenoxyacetic acid (2,4-D). Among the 2,4-D-degrading organisms belong, in particular, soil bacteria, such as, for example, Acinetobacter, Alcaligenes, Arthrobacter, Cyronebacterium and Pseudomonas [compare, in this connection, G. R. Bell, Can. J. Microbiol. 3 : 821 (1957); J. M. Bollag et al., J. Agric. Food Chem. 16 : 826 (1968); R. H. Don et al., J. Bacteriol. 145 : 681 (1981); W. C. Evans et al., Proc. Biochem. Soc. Biochem. J. 57 : 4 (1954); W. C. Evans et al., Biochem. J. 122: 543 (1971); R. P. Fisher et al., J. Bacteriol. 135 : 798 (1978); T. I. Steenson et al., J. Gen. Microbiol. 16 : 146 (1957); J. M. Tiedje et al., J. Agric. Food Chem. 17 : 1080 (1969); J. E. Tyler et al., Appl. Microbiol. 28 : 181 (1974); J. M. Tiedje et al., J. Agric. Food Chem. 17 : 1021 (1969)]. They play a significant part in the detoxification of the soil and of the wastewaters from halogenated aromatic compounds as they occur especially among the agriculturally utilized pesticides and herbicides.
In the group of the 2,4-D-degrading wild-type bacteria, the strain Alcaligenes eutrophus JMP134 is the most well-known and best-characterized. This strain harbors the plasmid pJP4 having a size of about 80 kilo-bases and containing all of the genes important for degradation of 2,4-D (R. H. Don et al., loc. cit.). The plasmid pJP4 has also been isolated and characterized recently [R. H. Don et al., J. Bacteriol. 161 : 466 (1985)].
Five genes participating in 2,4-D degradation, denoted as tfdB, tfdC, tfdD, tfdE and tfdF, were localized by transposon mutagenesis and cloned in E. coli. R. H. Don et al. [J. Bacteriol. 161 : 85 (1985)] succeeded in attributing enzyme function to four genes by biochemical studies. However, thus far attempts have been unsuccessful regarding localizing of gene tfdA on the plasmid pJP4 or on a subfragment of the latter, and to isolate this gene and to clarify its structure.